Calcium-independent negative regulation by CD81 of receptor signaling

ABSTRACT

Calcium independent CD81 inhibition of IgE-mediated degranulation in mast cells, particularly through the FcgammaRIII and FCepsiRI receptors, is described, as well as methods of inhibiting allergic processes.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 60/032,963, filed Dec. 13, 1996, the entire teachings of which areincorporated herein by reference.

GOVERNMENT FUNDING

Work described herein was funded by grant 1-RO1-GN53950-01 from theNational Institutes of Health. The U.S. Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

In the past two decades, tremendous advances have been made inunderstanding the molecular mechanisms used by various types of cellsurface receptors to transduce signals. Nearly all of these advanceshave come from the study of model systems where a receptor “activates”cells to generate a well-defined response. As knowledge about activatingmodel systems has increased, it has become clear that there are manysituations in which the activating signal sent from one receptor ismodulated as the direct result of a negative or inhibitory signal sentby another cell surface receptor. While the study of this type ofsignaling is generally in its infancy, several recent studies have begunto shed light on the molecular mechanisms which underliereceptor-mediated inhibitory signals in immunologic systems. Given thetendency of nature to utilize signaling functions modularly in a varietyof signaling pathways, the paradigms outlined by these systems may haveimplications for the study of inhibitory or deactivating signals innon-immunologic situations as well. In addition, the study of thesesignals may add new dimensions to the understanding of other widelyutilized signaling pathways.

SUMMARY OF THE INVENTION

As described herein, monoclonal antibodies (mAbs) have been isolatedwhich inhibit FcεRI-induced mast cell degranulation. Through proteinisolation, peptide sequencing, cloning, and gene expression, CD81 hasbeen identified as a novel inhibitory receptor for FcεRI and FcγRIII.Anti-CD81 mAbs also inhibited passive cutaneous anaphylaxis (PCA)reactions, a model of IgE-dependent, mast cell activation in vivo.

The invention pertains to a method of inhibiting cell surfacereceptor-mediated signaling comprising contacting a cell with an agentwhich induces CD81-mediated signal transduction. In a particularembodiment, the cell surface receptor is selected from the groupconsisting of FcεRI and FcγRIII. In one embodiment, the method is acalcium independent method.

The invention also relates to a method of inhibiting degranulationcomprising contacting a cell with an agent which induces CD81-mediatedsignal transduction. In one embodiment, degranulation is mediated by theFcεRI receptor. In another embodiment, the method is a calciumindependent method.

The invention further relates to a calcium independent method ofinhibiting cell surface receptor-mediated signaling in a mammal, such asa human, comprising administering to the mammal an effective amount ofan agent which induces CD81-mediated signal transduction. In oneembodiment, the cell surface receptor is selected from the groupconsisting of FcεRI and FcγRIII.

The invention also pertains to a method, e.g., a calcium independentmethod, of inhibiting degranulation induced by a cell surfacereceptor-mediated signal in a mammal, such as a human, comprisingadministering to the mammal an effective amount of an agent whichinduces CD81-mediated signal transduction.

The invention further pertains to a method of treating (e.g., preventingor reducing the severity of) an allergic condition in a mammal, such asa human, comprising administering to the mammal an effective amount ofan agent which induces CD81-mediated signal transduction. In particularembodiments, the allergic condition is asthma, hay fever or atopiceczema.

The invention also relates to a calcium independent method of enhancingcell surface receptor-mediated signaling, e.g., FcεRI-mediated signalingand FcγRIII-mediated signaling, comprising contacting a cell with anagent which inhibits CD81-mediated signal transduction.

The invention also pertains to a calcium-independent method of enhancingdegranulation comprising contacting a cell with an agent which inhibitsCD81-mediated signal transduction. For example, degranulation can bemediated by the FcεRI receptor. The invention also relates to a calciumindependent method of enhancing cell surface receptor-mediated signalingin a mammal comprising administering to the mammal an effective amountof an agent which inhibits CD81-mediated signal transduction.

The invention further relates to an assay for identifying agents whichalter CD81-mediated signal transduction, comprising combining a cellbearing CD81 with an agent to be tested, under conditions suitable forCD81-mediated signal transduction, and determining the level ofCD81-mediated signal transduction. If the level of CD81-mediated signaltransduction is altered relative to a control, the agent altersCD81-mediated signal transduction. In a particular embodiment, the agentis one which enhances or induces CD81-mediated signal transduction.

The invention also relates to an assay for identifying agents whichalter calcium independent CD81-mediated regulation of cell surfacereceptor signaling, comprising combining a cell bearing CD81 and anappropriate cell surface receptor with an agent which altersCD81-mediated signal transduction under conditions suitable for signaltransduction by CD81 and the cell surface receptor, and determining thelevel of cell surface receptor signaling. If the level of cell surfacereceptor signaling is altered relative to a control, the agent alterscalcium independent CD81-mediated regulation of cell surface receptorsignaling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate representative immunologic inhibitory signalingsystems. Solid dots on sIg, FcγRIII and TCR indicate tyrosinephosphorylation of activating motifs in the cytoplasmic tails of eachactivating receptor (FIGS. 1A-1C, respectively). Solid dots on FcγRIIB,KIR and CTLA-4 indicate tyrosine phosphorylation of inhibitory motifs inthe cytoplasmic tails of each inhibitory receptor (FIGS. 1A-1C,respectively). FIG. 1A illustrates the surface immunoglobulin receptor(sIg) complex and FcγRIIb1 system. FcγRIIb1 provides a negative feedbacksignal for soluble immunoglobulin production. FIG. 1B illustrates thenegative regulation of cytolytic immune cells by killer cell inhibitoryreceptors (KIR). FIG. 1C illustrates the negative regulation of T-cellreceptor-mediated activation signals by CTLA-4.

FIG. 2 illustrates the schematic structures of SHP1, SHP2 and SHIP.

FIG. 3 illustrates the proposed SHP and SHIP inhibitory signalingmechanisms. Solid dots on sIg indicate tyrosine phosphorylation ofactivating motifs in the cytoplasmic tails of each activating receptor.Solid dots indicate tyrosine phosphorylation of inhibitory motifs in thecytoplasmic tails of each inhibitory receptor.

FIG. 4 illustrates 5D1 mAb inhibition of FcεRI-mediated degranulation inRBL-2H3 cells.

FIG. 5 (SEQ ID NOS. 1-4) illustrates Ly-C peptide 1A12 sequence andalignment with mouse and human CD81.

FIGS. 6A-6B are the results of FACS analysis illustrating expression ofrat CD81 in CHO and NIH-3T3 cells. FIG. 6A shows stable expression ofrat CD81 in CHO cells stained with 1A12 mAb. FIG. 6B shows transientexpression of rat CD81 in NIH-3T3 cells infected with M.O.I.=5 of ratCD81 recombinant vaccinia virus and incubated for 6 hours prior tostaining with 5D1 mAb.

FIGS. 7A-7D are graphs of the effect of preincubatibn of purified mAb5D1 on FcεRI-mediated degranulation in RBL-2H3 cells. Data shownindicate the results of degranulation of IgE-saturated RBL-2H3 cellsafter incubation with buffer (filled circles) or purified 5D1 mAb at 2.5ng (filled squares), 25 ng (filled triangles), or 250 ng (filledinverted triangles) (FIGS. 7A, 7C, 7D) or with 100 ng (7B) of 5D1 mAbper 105 cells prior to triggering with the indicated concentrations ofDNP-HSA (FIG. 7A), 50 ng/ml DNP-HSA (FIGS. 7C and 7D) or with PMA andionomycin (FIG. 7B). Data are expressed as mean dpm±standard deviationor as percentages of control (no antibody) mean dpm. Statisticalsignificance versus untreated controls was determined using an unpairedStudent's t-test: *, p<0.05; **, p<0.01; ***, p<0.001 for FIG. 7A. Alldata points in FIGS. 7B and 7C were found to be significantly differentfrom controls (p<0.02) with the exception of the 5 minute preincubationtime point with 2.5 ng mAb 5D1 (FIG. 7C, p=0.067).

FIG. 8 shows expression of rat CD81 in mouse mast cell line C1.MC/C57.1by FACS staining with 5D1 and 1A12 mAbs.

FIGS. 9A-9C are graphs showing that CD81 mAbs fail to inhibitFcεRI-induced tyrosine phosphorylation, calcium mobilization, andleukotriene synthesis. FIG. 9A shows the effect of anti-CD81 on calciummobilization of fura-2-loaded RBL-2H3 cells triggered through FcεRI asmeasured by confocal microscopy. Fluo-3 fluorescence per ml ³Hmeasurements were normalized by dividing the average fluorescenceintensity (F) occurring during the course of the experiment to theaverage flourescence intensity at the beginning of the experiment (F₀)and expressed as F/F₀. Traces are shown of 10 individual cell (thinlines) together with mean values for these cells (thick lines) andrepresent typical results obtained from five separate experiments. FIG.9B shows ³H-serotonin release from RBL-2H3 cells prepared as in confocalmicroscopy measurements except that 3 μCi/ml ³H-serotonin was added tocultures. FIG. 9C shows LTC₄ measurements from 106 anti-DNP IgEsaturated RBL-2H3 treated with 1 μg 5D1 (open squares) or buffer (opencircles) prior to triggering with 30 ng/ml DNP-HSA for the indicatedperiods of time.

FIGS. 10A-10B are graphs showing inhibition of passive cutaneousanaphylaxis in Wistar rats by anti-CD81. Male Wistar rats were injectedwith (FIG. 10A) 25 ng DNP-specific IgE mixed with 50 μg anti-CD81 mAb5D1 (mouse IgG1) or control mouse IgG1 mAb (MOPC 31c, specificityunknown) or (FIG. 10B) 100 ng DNP-specific IgE alone. Statisticalsignificance was determined using an unpaired Student's t-test: *,p<0.05; **, p<0.01 (actual values 10A, p=0.024 versus MOPC 31c controls;10B, p=0.009 versus anti-LFA-10 controls).

FIGS. 11A-11D are the results of FACS analysis of 3 stable mouse FcγRIIIRBL-2H3 transfectants after staining with 2.4G2 and FITC-anti-rat IgG.

FIG. 12 is a set of graphs illustrating that DNP-HSA inducesIgE-mediated degranulation in four different cell lines and that thisdegranulation is inhibitable by anti-CD81 mAb 5D1.

DETAILED DESCRIPTION OF THE INVENTION

Mast cells are important effector cells in IgE-dependent immuneresponses and allergic diseases (Galli, New. Engl. J. Med. 328:257-265(1993)), and mast cells also contribute to host defense againstparasites and bacteria (Echtenacher et al., Nature 381:75-77 (1996);Galli and Wershil, Nature 381:21-22 (1996)). Crosslinking of FcεRI-IgEcomplexes on mast cells and basophils by multivalent antigen initiates asignaling cascade characterized by tyrosine kinase activation, calciumrelease and influx and, later, by degranulation and release ofinflammatory mediators (Jouvin et al., J. Biol. Chem. 269:5918-5925(1994); Penhallow et al., J. Biol. Chem. 270:23362-23365 (1995);Scharenberg et al., EMBO J. 14:3385-3394 (1995); Lin et al., Cell85:985-995 (1996); and (Paul et al., Adv. Imunol. 53:1-29 (1993)).

Like the B and T cell antigen receptors, FcεRI lacks endogenoussignaling capacity and utilizes tyrosine phosphorylation to recruitsignaling effector molecules. Receptor aggregation leads tophosphorylation and/or activation of several protein tyrosine kinases(PTKs) Lyn, Syk, Btk, Itk, Fer, and FAK (Jouvin et al., J. Biol. Chem.269:5918-5925 (1994); Penhallow et al., J. Biol. Chem. 270:23362-23365(1995); Scharenberg et al., EMBO J. 14:3385-3394 (1995); and Kawakami etal., Mol. Cell. Biol. 14:5108-5113 (1994); Kawakami et al., J. Immunol.155:3556-3562 (1995); and Hamawy et al., J. Biol. Chem. 268:6851-6854(1993)), as well as protein kinase C isoenzymes (Ozawa et al., J. Biol.Chem. 268:1749-1756 (1993)), MAP kinase (Hirasawa et al., J. Biol. Chem.270:10960-10967 (1995)), and other signaling molecules such as Cbl andShc (Ota et al., J. Exp. Med. 184:1713-1723 (1996); and Jabril-Cuenod etal., J. Biol. Chem. 271:16268-16272 (1996)).

The precise role of many of these proteins in degranulation remainsundefined. However, it is clear that FcεRI-mediated calciummobilization, degranulation, and leukotriene and cytokine synthesisdepend on early tyrosine kinase activation events, especially theactivation of the PTK Syk. FcεRI signaling is initiated by tyrosinephosphorylation of immunoreceptor tyrosine-based activation motifs(ITAM; defined by the sequence (D/E)x_(x)Yx₂Lx₆₋₇Yx₂(L/I) (Flaswinkel etal., Semin. Immunol 7:21-27 (1995)). Phosphorylated ITAMs (pITAMs)facilitate binding of SH2-domain-containing proteins to FcεRI (Johnsonet al., J. Immunol. 155:4596-4603 (1995); Kimura et al., J. Biol. Chem.271:27962-27968 (1996)).

In addition to activation events, receptor-activated PTKs initiate theregulation of antigen receptor signaling by phosphorylatingtyrosine-based motifs on membrane receptors known as inhibitoryreceptors (Scharenberg and Kinet, Cell 87:961-964 (1996); Cambier, Proc.Natl. Acad. Sci. USA 94:5993-5995 (1997)). These proteins bindSH2-domain-containing phosphatases, the tyrosine phosphatases SHP-1 andSHP-2 and the phosphatidylinositol (Scharenberg et al., EMBO J.14:3385-3394 (1995); Lin et al., Cell 85:985-995 (1996); Paul et al.,Adv. Immunol. 53:1-29 (1993)) 5′ phosphatase SHIP, upon coengagementwith antigen or growth factor receptors. Although the molecular targetsare still being defined, phosphatase recruitment to inhibitory receptorshas one of two general effects on signaling. Engagement of inhibitoryreceptors that preferentially bind SHIP, such as the low affinityreceptor for IgG (FcγRIIb1) (Ono et al., Nature 383:263-266 (1996)),results in selective inhibition of calcium influx with little or noeffect on receptor-mediated calcium release or tyrosine phosphorylation.On the other hand, killer cell inhibitory receptors (KIR) bind SHP-1upon receptor costimulation, resulting in reduced tyrosinephosphorylation, calcium release from the ER, and calcium influx(Burshtyn et al., Immunity 4:77-85 (1996); Binstadt et al., Immunity5:629-638 (1996)). In both mechanisms, calcium mobilization is inhibitedalong with downstream signaling events.

Descriptions of three representative systems utilized in recent studiesare useful for understanding the nature of inhibitory signals, and areoutlined in FIGS. 1A-1C. Briefly, the surface immunoglobulin receptor(sIg) complex and FcγRIIb1 (a low affinity receptor for IgG) are bothnormally present on B-cell surfaces (FIG. 1A, left panel). When sIgreceptors are clustered as a result of contact with antigen (FIG. 1A,middle panel), they typically produce a cell activation signal whichinduces B-cell proliferation. However, if the same B-cells arestimulated so that the sIg receptors are co-clustered with FcγRIIb1receptors (for example by contact of the B-cell with an immune complexof cognate antigen and IgG, FIG. 1A, right panel), B-cells fail toproliferate and in some cases may apoptose.

In the natural killer (NK) cell system, a number of cell surfacereceptors are able to initiate NK cell cytolysis, one of which isFcγRIII (FIG. 1B, left panel). When an NK cell encounters a target cell,it recognizes and kills the target cell if the target cell lacks class IMHC molecules. One of the ways in which NK cells recognize target cellsis by binding of IgG bound to the target cell surface to FcγRIII onNK-cells (FIG. 1B, middle panel). If the target cells expressappropriate class I MHC molecules which can be recognized by appropriatekiller cell inhibitory receptors (KIR) on the NK cell, they areprotected from cytolysis (FIG. 1C, right panel).

In the T-cell system, the T-cell antigen receptor (TCR) and CD4 and/orCD8 co-receptors are normally expressed on the surface of restingT-cells (FIG. 1C, left panel). T-cells are activated when their T-cellantigen receptor complexes (TCR's) interact with specific peptide/MHCclass II complexes on antigen presenting cells (APCs), resulting inco-clustering of the TCR and CD4 or CD8 (FIG. 1C, middle panel). Uponactivation, T-lymphocytes upregulate expression of another surfacemolecule called CTLA-4, which results in interaction of CTLA-4 with itscountereceptors CD80 or CD86 (FIG. 1C, right panel). Since mice whichlack CTLA-4 have hyperactivated T-cells and are prone tolymphoproliferative diseases, it is thought that CTLA-4 mediates aninhibitory signal which provides an important negative feedback controlfor proliferation and cytokine production induced by T-cell receptoractivation signals.

While each of these systems is unique in terms of the manner in whichthe activating and inhibitory signals are engaged, two common featuresexist among them: 1) Each involves activating signals mediated byhomologous cytoplasmic tail motifs known as immunoreceptor tyrosinebased activation motifs (ITAMs). These motifs become tyrosinephosphorylated by src family kinases when the activating receptors areengaged by clustering stimuli, resulting in the recruitment to engagedreceptors of both src and syk/zap70 family non-receptor tyrosinekinases. Downstream propagation of the activation signal is thenmediated by activation of these tyrosine kinases and the resultingphosphorylation of specific substrates. 2) The inhibitory signals aremediated by separate receptors, such as FcγRIIb1, killer cell inhibitoryreceptors (KIR), and CTLA-4, which are engaged in concert with theactivating receptor when an appropriate stimulus is present. When theinhibitory receptors are appropriately engaged, they becomephosphorylated on specific cytoplasmic tail tyrosines by src familykinases, which results in the recruitment of signaling molecules whichare inhibitory in function.

It appears that SHP-1/SHP-2 and SHIP are recruited for distinctpurposes. SHP-1 and SHP-2 attenuate or completely block tyrosinephosphorylation-mediated signals (FIG. 3, middle panel), while SHIPallows a full strength tyrosine phosphorylation signal to proceed whileblocking any downstream events which require sustained elevations ofsoluble inositol phosphates and/or intracellular calcium (FIG. 3, rightpanel). One potential explanation can be rationalized by comparing thefunction of the inhibitory signals mediated by FcγRIIb1 on B-cells andKIR on NK cells. The sIg receptor activating signal serves to notifyB-cells that specific antigen is present, and so initiate B-cellmaturation and proliferation for the purpose of specific immunoglobulinproduction. However, coengagement of sIg and FcγRIIb1 blocksproliferation and can induce apoptosis of the B-cell and a consequentdecrease in production of specific immunoglobulin, thereby acting as anegative feedback mechanism. Thus, it appears that the persistence of afull strength tyrosine phosphorylation signal in the absence ofsustained inositol phosphate and/or intracellular calcium levels is forthe purpose of notifying the B-cell that adequate specific antibody hasbeen produced, and may be the signal which induces apoptosis of thatB-cell in the appropriate context.

This situation is subtly, but importantly, different than that of an NKcell. NK cells function by undergoing target cell recognition eventsmediated by activating receptors which are capable of initiatingcytolysis, and the KIR inhibitory signal is required to blockinappropriate cytolysis of cells which are recognized but which alsobear appropriate class I MHC. Since there would be little utility in theNK cell “knowing” about contact with each and every protected target, aninhibitory mechanism where the activating signal is completely abrogatedwould seem to be most appropriate. This would account for the apparentlySHP-1 predominant inhibitory signal mediated by KIR. To summarize, theseresults suggest that primarily SHP-1/SHP-2 mediated block would beutilized when the cell has no need to know about the presence of aparticular stimulus, while a primarily SHIP-mediated block would beutilized when the cell needs to know and to respond in some alteredmanner.

IgE-dependent activation of mast cells primarily occurs throughantigen-mediated crosslinking of IgE-FcεRI complexes which initiates asignaling cascade ultimately leading to release of proinflammatorymediators (Scharenberg and Kinet, Chem. Immmunol. 61:72-87 (1995)).FcεRI is a member of the multi-subunit, antigen receptor family whichincludes B and T cell receptors (BCR and TCR) and receptors for the Fcportions of IgA and IgG (Ravetch and Kinet, Ann. Rev. Immunol. 9:457-492(1991)). These receptors share common features of immunoglobulin-likeligand binding subunit(s) and associated signaling polypeptides whichlack endogenous enzymatic activity.

In mast cells, both FcεRI and FcγRIII are expressed as αβγg_(γ2)tetramers in which the respective β and FcRγ signaling chains areidentical and the ligand-binding α chains are different. In FcεRI, thehigh affinity IgE binding domain is localized to the FcεRIα subunit(Blank et al., J. Biol. Chem. 266:2639-2646 (1991)) and IgE binding toFcεRIβ itself does not contribute to signaling. The FcεRIβ chain and theFcRg homodimer are the signaling components of the FcεRI (αβγ2)tetrameric receptor. Both FcεRIβ and FcRγ have one copy per chain of theimmunoreceptor tyrosine-based activation motif (ITAM; Flaswinkel et al.,Semin. Immunol. 7:21-27 (1995), Cambier, J. Immunol. 155:3281-3285(1995)) defined by the sequence Y×2L×6-7Y×2L/I.

FcεRI signaling is an aggregation-dependent phenomenon in whichmultivalent antigen crosslinking of IgE-FcεRI complexes initiates asignaling cascade ITAM tyrosine phosphorylation by src family kinases(Shaw et al., Semin. Immunol. 7:13-20 (1995)). Signaling through FcεRIis characterized initially by tyrosine phosphorylation of FcεRIβ andFcRγ ITAMs by the β-associated src family kinase lyn (Jouvin et al., J.Biol. Chem. 269:5918-5925 (1994)). The lyn-phosphorylated ITAM (pITAM)interaction results in lyn activation. Direct binding of lyn to fusionproteins containing the FcεRIβ, but not the FcRγ ITAM, has beendemonstrated (Jouvin et al., J. Biol. Chem. 269:5918-5925 (1994)). pITAMpeptides have been shown to induce lyn phosphorylation both inpermeabilized cells and in vitro (Johnson et al., J. Immunol.155:4596-4603 (1995)).

Following lyn activation, syk is recruited to FcRγ pITAMs via its SH2domains where it is phosphorylated and activated (Scharenberg and Kinet,Chem. Immunol. 61:72-87 (1995); Jouvin et al., J. Biol. Chem.269:5918-5925 (1994)). FcRγ PITAM peptides were much more effective thanFcεRIβ pITAM peptides at activating syk in vitro in unstimulated RBL-2H3lysates (Shiue et al., J. Biol. Chem. 270:10498-10502 (1995)). Activatedlyn and syk phosphorylate a number of intracellular substrates includingPLCγ1, BTK, ITK and cbl (Rawlings et al., Science 271:822-825 (1996);Kawakami et al., J. Immunol. 155:3556-3562 (1995)). Following initialtyrosine kinase activation events, FcεRI signaling, like that of otherantigen receptors, involves calcium release from the endoplasmicreticulum (tyrosine kinase-dependent) and a calcium influx, both ofwhich precede degranulation and the release of preformed mediators bygranule fusion with the cytoplasmic membrane. An interesting differencebetween FcεRI and other antigen receptors is that calcium mobilizationthrough FcεRI appears to utilize sphingosine kinase andsphingosine-1-phosphate (S-1-P) (Choi et al., Nature 380:634-636 (1996))as opposed to the classical phospholipase C/InsP3 pathway.

The rat basophilic leukemia cell line, RBL-2H3, has been widely employedas a model cell in the study of FcεRI-mediated activation. There havebeen a few reports of monoclonal antibodies (mabs) directed to membranecomponents in which co-ligation inhibits FcεRI-mediated degranulation inmast cells. The best characterized examples are MAFA (mast cellfunction-associated antigen) (Guthmann et al., Proc. Natl. Acad. Sci.USA 92:9397-9401 (1995)) and gp49b1 (Katz et al., Proc. Natl. Acad. Sci.USA 93:10809-10814 (1996)). MAFA is an Mr 20 kd C-type lectin expressedin RBL-2H3 cells both as a monomer and disulphide-linked homodimer thatinhibits degranulation by acting upstream of FcεRI-mediated activationof phospholipase Cg1 activation by tyrosine kinases (Guthmann et al.,Proc. Natl. Acad. Sci. USA 92:9397-9401 (1995)).

The target of gp49B1 is less well defined; however it appears to act viaa tyrosine-based ITIM (immunoreceptor tyrosine-based inhibitory motif)defined by the sequence V/I×2Y×2I/L utilized by the NK inhibitoryreceptor (KIR), CD22, CTLA-4, and FcγRIIβ1. Tyrosine phosphorylation ofthe ITIM in KIR induces binding of the SHP-1 tyrosine phosphatase. SHP-1recruitment is intimately associated with inhibition of calcium influxand mobilization presumably enacted through yet-to-defineddephosphorylation events. Overexpression of phosphatase-inactive SHP-1ablates the inhibitory activity of endogenous SHP-1. ITIM-mediatedrecruitment is not restricted to SHP-1, as a second SH2-containingphosphatase (SHP-2) is utilized by CTLA-4, and the FcgRIIb1 ITIM bindseither SHP-1 or the SH2-containing inositol phosphatase (SHIP). In thecase of gp49b1, it is unclear which effector is being utilized but ithas been demonstrated that a splice variant (gp49A) which lacks thecytoplasmic ITIM but is identical in the extracellular domains lacksdetectable inhibitory activity. In addition to MAFA, antibodies to theglycolipid Gd1b and the AD1 antigen (rat homologue of CD63) have alsobeen described to inhibit FcεRI-mediated degranulation in RBL-2H3 cells.

Clustering of the high affinity IgE receptor (FcεRI) by antigeninitiates a signaling cascade characterized by tyrosine kinaseactivation, calcium release and influx and, later, by degranulation andrelease of inflammatory mediators. In order to examine how FcεRIsignaling is negatively regulated, a panel of monoclonal antibodies tomast cell membrane antigens was generated and screened for inhibition ofIgE-mediated mast cell degranulation. Two degranulation inhibitoryantibodies, designated 1A12 and 5D1, immunoprecipitated a Mr 25 kdprotein from surface-iodinated rat basophilic leukemia (RBL-2H3) cells.Lys-C peptide sequence obtained from 1A12-immunoaffinity purifiedimmunoprecipitates was found to be highly homologous to mouse and humanCD81. Subsequent cloning and expression of rat CD81 cDNA from RBL-2H3confirmed that 1A12 and 5D1 recognize rat CD81 and that CD81crosslinking inhibits FcεRI-mediated mast cell degranulation.

Signaling through the high affinity receptor for immunoglobulin E(FcεRI) results in the coordinate activation of tyrosine kinases priorto calcium mobilization. Receptors capable of interfering with thesignaling of antigen receptors, such as FcεRI, recruit tyrosine andinositol phosphatases that results in diminished calcium mobilization.It is shown herein that antibodies recognizing CD81 inhibitFcεRI-mediated mast cell degranulation but, surprisingly, withoutaffecting aggregation-dependent tyrosine phosphorylation, calciummobilization, or leukotriene synthesis. Furthermore, CD81 antibodiesalso inhibit mast cell degranulation in vivo as measured by reducedpassive cutaneous anaphylaxis responses. These results reveal anunsuspected calcium-independent pathway of antigen receptor regulationwhich is accessible to engagement by membrane proteins and on whichnovel therapeutic approaches to allergic diseases can be based.

CD81 belongs to the transmembrane 4 superfamily (TM4SF) which includesCD9, CD53, CD63 and CD82 (Wright and Tomlinson, Immunol. Today15:588-594 (1994)). TM4SF proteins have been found to associate withHLA-DR, CD4, CD19/21/Leu-13, small GTP-binding proteins and anunidentified tyrosine phosphatase and (via mAb crosslinking) to inducecalcium mobilization and activate syk.

CD81 is broadly expressed on hematopoietic cells (T and B lymphocytes,granulocytes, monocytes) and on some non-lymphoid tumors. The functionof CD81 (or other TM4SF proteins) is incompletely understood, althoughCD81 appears to modulate the signaling of other membrane receptors. CD81is found in the CD19/CD21 complex on B cells, and mAbs to CD81 or CD19have been reported to reduce the threshold for B cell receptor signaling(Fearon and Carter, Annu. Rev. Immunol. 13:127-149 (1995)) and enhance Bcell adhesion via VLA4 (Behr and Schriever, J. Exp. Med. 182:1191-1199(1995)). Consistent with a costimulatory role in B cell receptorsignaling, CD81 −/− mice express lower levels of CD19 on B cells whichis proposed to contribute to a defect in humoral immunity (Maecker andLevy, J. Exp. Med. 185:1505-1510 (1997)). For T lineage cells, bothstimulatory and inhibitory activities for anti-CD81 mabs have beenreported (Secrist et al., Eur. J. Immunol. 26:1435-1442 (1996); Todd etal., J. Exp. Med. 184:2055-2060 (1996); Oren et al., Mol. Cell. Biol.10:4007-4015 (1990); and Boismenu et al., Science 271:198-200 (1996)).CD81 ligation enhances IL-4 production from antigen-specific CD4+ Tcells (Secrist et al., Eur. J. Immunol. 26:1435-1442 (1996)) andintegrin activation and IL 2-dependent proliferation in human thymocytes(Todd et al., J. Exp. Med. 184:2055-2060 (1996)). Alternatively, CD81was originally called TAPA-1 (target of antiproliferative antibody)based on inhibition of proliferation in human B cell lines by CD81antibodies (Oren et al., Mol. Cell. Biol. 10:4007-4015 (1990)). Some ofthese pleiotropic effects may stem from the potential signalingmolecules with which CD81 has been reported to associate including CD4,CD8, MHC class II, other TM4SF proteins, integrin VLA4, andphosphatidylinositol 4-kinase (Wright and Tomlinson, Immunol. Today15:588-594 (1994); Imai et al., J. Immunol. 155:1229-1239 (1995);Angelisova et al., Immunogenetics 39:249-256 (1994); Mannion et al., J.Immunol. 157:2039-2047 (1996); and Berditchevski et al., J. Biol. Chem.272:2595-2598 (1997)).

Mast cell FcεRI can be saturated with monoclonal IgE antibodies. In theabsence of crosslinking by appropriate antigen, IgE binding to FcεRIdoes not activate mast cells. Monoclonal antibodies are purified fromculture supernatants or mouse ascitic fluid (produced by injection ofantibody-producing cells into immunocompromised mice by standardtechniques, such as those described in Kohler and Milstein, Nature256:495-497 (1975); Kozbar et al., Immunology Today 4:72 (1983); andCole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96 (1985)). Crosslinking by the antigen (protein binding tothe IgE) normally induces cell degranulation which can be quantitated byenzyme assay or radioactivity release assay. Antibody treatment of CD81mast cells inhibits IgE-mediated degranulation; 20 ng of 5D1 monoclonalantibody per 10₅ RBL-2H3 cells inhibits degranulation throughIgE-mediated channels by greater than 75%.

Mast cells are a major cell in allergic reactions. Thus, the presentinvention can be used to develop agents, e.g., antibodies, which inhibitthe allergic process, as well as to develop compounds for the treatmentof allergies, anaphylactic reactions and related diseases. Agents canalso be developed which mimic the process of CD81-mediated inhibition ofmast cell degranulation. Anti-CD81 antibodies are more inhibitory thanantibodies to other different proteins for IgE-mediated degranulation,particularly because anti-CD81 antibodies act directly and do notrequire secondary reagents. The work described herein can also be usedto develop model systems for the study of activation of mast cellsthrough the FcεRI receptor and to improve the therapeutic capability tomodulate the function of these cells.

Agents described herein can be anything which binds to or interacts withCD81 and induces (i.e., activates) or enhances CD81-mediated signaltransduction. For example, the agent can be a small molecule, a peptide,or a polyclonal or monoclonal antibody, such as an anti-CD81 antibody.In particular embodiments, the antibody is 5D1 or 1A12.

In order to identify membrane proteins capable of regulating FcεRIsignaling, mAbs to the rat basophilic leukemia (RBL-2H3) cell line wereproduced and antibodies which inhibited FcεRI-mediated degranulationwere identified. The results are shown in FIGS. 7A-7D. Cells werepreincubated with mAb 5D1 or buffer for 30 minutes (FIGS. 7A, 7B, 7D) orfor the indicated times (FIG. 7C) at room temperature prior totriggering for 30 minutes (FIGS. 7A-7C) or as indicated (FIG. 7D). Thedata shown are representative of more than 10 experiments with the 5D1mAb. As shown in FIG. 7A, pretreatment of anti-DNP IgE-saturated RBL-2H3cells with purified mAb 5D1 inhibited FcεRI-mediated degranulation by75% as measured by release of granule-stored ³H-serotonin. Blockage ofserotonin release was significant (*, p<0.05) even at subsaturatingconcentrations of 5D1 (2.5 nm mAb/10⁵ cells, FIG. 7A). 5D1-mediatedinhibition was specific for FcεRI signaling, as degranulation induced byphorbol myristate acetate (PMA) and calcium ionophore ionomycin wereunaffected (FIG. 7B). Furthermore, maximal inhibition of FcεRI-mediateddegranulation by mAb 5D1 required only brief periods of preincubation(FIG. 7C), and inhibition was sustained for at least one hour of antigenstimulation (FIG. 7D).

The protein recognized by the degranulation-inhibitory 5D1 mAb was thenidentified. 5D1 and a second degranulation-inhibitory mAb (1A12)recognized proteins of Mr 25 kDa. 5D1 and 1A12 blocked each others'binding to RBL-2H3 cells, although neither mAb inhibited IgE bindingand, conversely saturation of FcεRI with IgE had no effect on 1A12binding, suggesting that 1A12 and 5D1 recognized the same protein (seeFIG. 8) and that FcεRI and the 1A12/5D1 antigen were not co-localized onthe cell membrane. Since mAb 1A12 was more effective atimmunoprecipitation and on Western blots, it was used for proteinpurification. Batch preparations of RBL-2H3 extracts wereimmunoprecipitated with mAb 1A12, resolved on preparative SDS-PAGE andtransferred to nitrocellulose for protein sequencing. Peptide sequenceobtained from Lys-C digests of 1A12 immunoprecipitates is shown alignedwith homologous sequences from mouse and human CD81 in FIG. 5. Based onthese data, rat CD81 was cloned from a RBL-2H3 cDNA library using mouseCD81 cDNA as a probe and expressed in the mouse mast cell lineC1.MC/C57.1 (Young et al., Proc. Natl. Acad, Sci. USA 84:9175-9179(1987)). FACS profiles of C1.MC/C57.1 transfectants are shown in FIG. 8;both degranulation-inhibitory mAbs 1A12 and 5D1 recognized rat CD81.

To target the site of CD81 inhibition of degranulation, the effect ofCD81 antibodies on the earliest events of FcεRI signal transduction,i.e. tyrosine phosphorylation of proteins by activated, nonreceptortyrosine kinases including Lyn and Syk, and calcium mobilization (Jouvinet al., J. Biol. Chem. 269:5918-5925 (1994); Penhallow et al., J. Biol.Chem. 270:23362-23365 (1995); Scharenberg et al., EMBO J. 14:3385-3394(1995); Lin et al., Cell 85:985-995 (1996)) was examined. In theseexperiments, IgE-saturated RBL-2H3 cells were pretreated with purifiedanti-CD81 prior to triggering with DNP-HSA for the indicated periods oftime, followed by extraction and immunoprecipitation of totaltyrosine-phosphorylated proteins. No major changes in the pattern ofFcεRI-induced tyrosine phosphorylation were detected with anti-CD81treatment prior to antigen triggering. Incubation of RBL-2H3 cells with5D1 alone (no antigen triggering) did not induce detectable tyrosinephosphorylation.

The effect of anti-CD81 on FcεRI-induced calcium mobilization wasmonitored on individual, adherent RBL-2H3 cells by confocal microscopyin cells loaded with calcium dye fluo-3. As shown in FIG. 9A, noinhibition of FcεRI-induced calcium mobilization in anti-CD81 treatedversus controls was observed by confocal microscopy, despite inhibitionof degranulation under these conditions (FIG. 9B). Anti-CD81pretreatment had no effect on calcium release from intracellular storesin cells triggered in Ca²⁺-free buffer containing 0.5 mM EGTA or onpre-triggering baseline values. Similar results were also obtained withRBL-2H3 triggered through FcεRI in suspension using a spectrophotometer.In separate experiments, anti-CD81 mAb 5D1 did not inhibit leukotrieneC₄ (LTC₄) production induced by DNP-HSA/IgE stimulation (FIG. 9C). LTC4production is dependent on activation of phospholipase A2 (tyrosinekinase and calcium-dependent) and is regulated by PMA-sensitive, proteinkinase C isozymes (Currie et al., Biochem. J. 304:923-928 (1994)); Aliet al., J. Immunol. 153:776-788 (1994)). These data suggest that CD81acts independently of early tyrosine phosphorylation and calciummobilization events which are critical for mast cell degranulation.

These results were unexpected in light of the reported modes of actionof other inhibitory receptors. These proteins fall into two majorclasses; type I, transmembrane proteins that are members of the Igsuperfamily (FcγRIIb1, KIR, CTLA-4, CD22, gp49b1, paired Ig-likereceptors (PIR), signal-regulatory proteins (SIRPs)) and type II,transmembrane, C-type lectins (e.g. Ly-49, NKG2A, mast cell functionassociated protein (MAFA)) (Ono et al., Nature 383:263-266 1996);Burshtyn et al., Immunity 4:77-85 (1996)).

CD81 differs from these inhibitory receptors in three important ways.First, unlike other inhibitory receptors, CD81 inhibits FcεRI-mediateddegranulation while leaving both tyrosine phosphorylation and calciummobilization apparently unaffected. While these results cannot exclude avery selective inhibition of kinase activity by CD81 antibodies, it isclear that no detectable effect is found on tyrosine kinase-sensitivecalcium mobilization of LTC₄ production. Second, CD81 belongs to adifferent structural class of proteins than the other inhibitoryreceptors. CD81 is a TM4SF protein with four transmembrane spanningsegments, two extracellular loops, two short cytoplasmic tails, and ashort intracellular loop between transmembrane segments 2 and 3 (Wrightand Tomlinson, Immunol. Today 15:588-594 (1994)). Third, the cytoplasmictails of CD81 lack ITIM motifs. While there is an ITIM-like sequence(GCYGAI) in the short intracellular loop between transmembrane segments2 and 3, there is no evidence that this site is phosphorylated bytyrosine kinases or capable of binding to SH2 domains.

In order to assess the activity of anti-CD81 in FcεRI signaling innormal mast cells, the passive cutaneous anaphylaxis (PCA) model, aclassic system for studying mast cell activation in vivo (Wershil etal., J. Immunol. 154:1391-1398 (1995); Dombrowicz et al., J. Clin.Invest. 99:915-925 (1997)), was chosen. In these experiments, rats wereinjected intradermally with IgE mixed with anti-CD81 mAb 5D1 (IgG1) orwith class-matched mouse (IgG1) as control (FIG. 10A). Additional ratsreceived anti-DNP IgE alone into the skin at time 0, followed by asecond injection (buffer, 5D1, or anti-rat LFA-1β (IgG1)) (FIG. 10B)into IgE-injected sites 21 hours after IgE injections. Twenty four hoursafter IgE priming, rats received 1 mg of antigen intravenously (DNP-HSAcontaining 1% Evan's blue dye). Mast cell activation through FcεRI inPCA results in the release of several vasoactive substances which act toincrease vascular permeability, a property which is quantified by localaccumulation of the Evan's blue dye from the vasculature into the sitesof IgE injections. These results are expressed as μg Evan's blueconverted from A₆₁₀ measurements of formamide-extracted tissue biopsies(Dombrowicz et al., J. Clin. Invest. 99:915-925 (1997)). As shown inFIG. 10A, coinjection of anti-CD81 mAb 5D1 during IgE primingsignificantly inhibited IgE-dependent PCA reactions (p=0.024) comparedto class-matched controls.

To limit the possibility of non-specific suppression of PCA reactionsdue to tissue deposition of IgG₁ mAbs, these experiments were repeatedby injecting anti-CD81 mAb 5D1 or anti-LFA-1β (CD18) into theIgE-injected sites 3 hours before antigen administration. LFA-1β isexpressed on mast cell lines including RBL-2H3 but anti-LFA-1β has noeffect on FcεRI-mediated degranulation in RBL-2H3 cells (Weber et al.,Scand. J. Immunol. 45:471-481 (1997)). Similar to coinjection of IgE andIgG₁ mAbs, separate injections of anti-CD81 yielded significantinhibition of PCA reactions compared to anti-LFA-1β controls (FIG. 10B).

Thus, it is demonstrated herein that CD81 is a novel inhibitory receptorfor FcεRI. The observation that CD81 acts on calcium-independent eventsrequired for mast cell degranulation distinguishes CD81 from previouslydescribed inhibitory receptors, such as FcγRIIb1 and KIR, which actupstream of calcium influx. Anti-CD81 mAbs also inhibited IgE-dependentPCA reactions, which suggests the CD81 pathway is present in normal mastcells and capable of being engaged to inhibit mast cell responses invivo. Therefore, the CD81 inhibitory pathway can be utilized intherapeutic strategies aimed at intervention of allergic responses.

RBL-2H3 cells express FcεRI, CD81 and endogenous rat FcγRIII receptors.However, no high-affinity reagent (antibody) is available to trigger theFcγRIII receptors on RBL-2H3; the 2.4G2 antibody (anti-mouseFcγRII/FcγRIII) was used for this purpose. To demonstrate that CD81stimulation inhibits degranulation induced through FcγRIII signaling asit does for FcεRI, murine FcγRIIIα chain cDNA was expressed in RBL-2H3cells.

FcγRIII binding of IgG is detectable only when IgG is present in theform of IgG-containing immune complexes which crosslink FcγRIIIreceptors and initiate intracellular signals. One of the methods oftriggering FcγRIII is through stimulation with crosslinked anti-FcγRIIIantibodies. FIG. 12 shows the results when RBL-2H3 andFcγRIII-transfectants of RBL-2H3 were loaded with ³H-serotonin in thepresence (DNP-HSA stimulation) or absence (immune complex stimulation)of DNP-specific IgE. After overnight incubation, cells were washed andincubated with culture media or media containing 200 ng of anti-rat CD81mAb 5D1 prior to triggering with optimized concentrations of DNP-HSA orwith preformed immune complexes of 2.4G2/anti-rat IgG F(ab′)₂.Degranulation was allowed to proceed for 30 minutes at 37° C. andreleased ³H-serotonin was quantitated by scintillation counting. Asshown in FIG. 12, DNP-HSA induces IgE-mediated degranulation in all fourcell lines which is inhibitable by anti-CD81 mAb 5D1. 2.4G2/anti-rat IgGF(ab′)2 preformed complexes, but not anti-rat IgG F(ab)2 alone, inducedegranulation only in cells expressing mFcγRIII receptors (RBL-2H3transfectants A10, D10 and H11), a process which is also inhibitable bypreincubation with 5D1. This data provides the identification of CD81 asa common inhibitor of both FcεRI and FcγRIII.

Accordingly, the present invention relates to a method of inhibiting orenhancing cell surface receptor signaling, e.g., FcεRI-mediated orFcγRIII-mediated signaling. The method of inhibiting cell surfacereceptor signaling comprises contacting a cell with an effective amountof an agent which enhances or induces CD81-mediated signal transduction.Alternatively, the method can be a method of inhibiting cell surfacereceptor signaling in a mammal, comprising administering to the mammalan effective amount of an agent which enhances or induces CD81-mediatedsignal transduction. Appropriate cells are any cell type which expressesor has been designed to express (e.g., by transfection or geneticengineering) both CD81 and a suitable cell surface receptor.

For example, inhibition of the cell surface receptor signals whichinduce mast cell degranulation is useful in methods of treating allergicconditions or inflammatory disorders. Enhancement of the cell surfacereceptors which induce mast cell degranulation is useful in inducing aninflammatory response, for example, in response to bacterial or parasiteinfection.

The method of enhancing cell surface receptor signaling comprisescontacting a cell with an effective amount of an agent which inhibits orprevents CD81-mediated signal transduction. Alternatively, the methodcan be a method of enhancing cell surface receptor signaling in amammal, comprising administering to the mammal an effective amount of anagent which inhibits or prevents CD81-mediated signal transduction. Itmay be clinically beneficial to enhance cell surface receptor signalingin a mammal, or the functional results thereof, such as degranulation,in conditions where an inflammatory response and/or release ofleukotrienes and cytokines is beneficial, such as in host defenseagainst parasites and bacteria.

The invention also pertains to a method of treating an allergy (e.g.,asthma, hay fever or atopic eczema) or inflammatory condition in amammal comprising adminsitering to the mammal an effective amount of anagent which induces CD81-mediated signal transduction. For example, themethod can be used to treat allergic or inflammatory responsesassociated with disorders such as autoimmune (Type I) diabetes mellitus,rheumatoid arthritis, ankylosing spondylitis, sarcoidosis, Sjögren'ssyndrome, multiple sclerosis, inflammatory bowel disease (i.e., Crohn'sdisease and ulcerative colitis), dermatomyositis, scleroderma,polymyositis, systemic lupus erythematosus, biliary cirrhosis,autoimmune thyroiditis, and autoimmune hepatitis, as well as manydermatological disorders, including psoriasis, contact sensitivity andatopic dermatitis.

As used herein, “inhibit” is intended to encompass any qualitative orquantitative reduction in a measured effect or characteristic, includingcomplete prevention, relative to a control. As also used herein,“enhance” is intended to encompass any qualitative or quantitativeincrease in a measured effect or characteristic relative to a control.An “effective amount” of a given agent is intended to mean an amountsufficient to achieve the desired effect, e.g., the desired therapeuticeffect, under the conditions of administration, such as an amountsufficient for inhibition or enhancement of CD81-mediated signaltransduction.

The present invention also relates to preparations for use in theinhibition or enhancement of cell surface receptor signaling, and thetreatment of allergic diseases and inflammatory disorders, thepreparation including an inhibitor or promoter of CD81-mediated signaltransduction, together with a physiologically acceptable carrier andoptionally other physiologically acceptable adjuvants.

According to the method, a therapeutically effective amount of one ormore agents (e.g., a preparation comprising an inhibitor or promoter ofCD81-mediated signal transduction can be administered to an individualby an appropriate route, either alone or in combination with anotherdrug.

A variety of routes of administration are possible including, but notlimited to, oral, dietary, topical, parenteral (e.g., intravenous,intraarterial, intramuscular, subcutaneous injection), and inhalation(e.g., intrabronchial, intranasal or oral inhalation, intranasal drops)routes of administration, depending on the agent and disease orcondition to be treated. For respiratory allergic diseases such asasthma, inhalation is a preferred mode of administration.

Formulation of an agent to be administered will vary according to theroute of administration selected (e.g., solution, emulsion, capsule). Anappropriate composition comprising the agent to be administered can beprepared in a physiologically acceptable vehicle or carrier. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils, for instance. Intravenous vehicles caninclude various additives, preservatives, or fluid, nutrient orelectrolyte replenishers and the like (See, generally, Remington'sPharmaceutical Sciences, 17th Edition, Mack Publishing Co., PA, 1985).For inhalation, the agent can be solubilized and loaded into a suitabledispenser for administration (e.g., an atomizer, nebulizer orpressurized aerosol dispenser).

Furthermore, where the agent is a protein or peptide, the agent can beadministered via in vivo expression of the recombinant protein. In vivoexpression can be accomplished via somatic cell expression according tosuitable methods (see, e.g. U.S. Pat. No. 5,399,346). In thisembodiment, nucleic acid encoding the protein can be incorporated into aretroviral, adenoviral or other suitable vector (preferably, areplication deficient infectious vector) for delivery, or can beintroduced into a transfected or transformed host cell capable ofexpressing the protein for delivery. In the latter embodiment, the cellscan be implanted (alone or in a barrier device), injected or otherwiseintroduced in an amount effective to express the protein in atherapeutically effective amount.

The invention also pertains to assays for identifying agents whichenhance or inhibit calcium independent CD81-mediated signaltransduction. The assay comprises combining a cell bearing CD81 with anagent to be tested, under conditions suitable for signal transduction byCD81. The level or extent of CD81-mediated signal transduction can bemeasured using standard methods and compared with the level or extent ofCD81-mediated signal transduction in the absence of the agent (control).An increase in the level or extent of CD81-mediated signal transductionrelative to the control indicates that the agent is a promoter ofCD81-mediated signal transduction; a decrease in the level or extent ofCD81-mediated signal transduction relative to the control indicates thatthe agent is an inhibitor of CD81-mediated signal transduction.

Inhibitors or promoters of CD81-mediated signal transduction, e.g.,those identified by methods described herein, can be assessed todetermine their effect on cell surface receptor signaling. Inhibitors orpromoters of CD81-mediated regulation of cell surface receptor signalingcan be, for example, small molecules, antibodies and/or peptides. A cellbearing CD81 and an appropriate cell surface receptor (e.g., FcεRI orFcγRIII) are combined with an inhibitor or promoter of CD81-mediatedsignal transduction under conditions suitable for signal transduction byboth CD81 and the cell surface receptor. The level or extent of cellsurface receptor signaling can be measured using standard methods andcompared with the level or extent of cell surface receptor signaling inthe absence of the inhibitor or promoter (control). An increase in thelevel or extent of cell surface receptor signaling relative to thecontrol indicates that the agent is a promoter of cell surface receptorsignaling; a decrease in the level or extent of cell surface receptorsignaling relative to the control indicates that the agent is aninhibitor of cell surface receptor signaling.

Cell surface receptor signaling can be measured directly, such as bymeasuring the level or amount of an associated signalling molecule, orindirectly, such as by a functional assay measuring level or amount ofdegranulation or passive cutaneous anaphylaxis.

The following Examples are offered for the purpose of illustrating thepresent invention and are not to be construed to limit the scope of thisinvention. The teachings of all references cited herein are herebyincorporated herein by reference.

EXAMPLES Cell Culture, Reagents and Antibodies

The rat basophilic leukemia cell line (RBL-2H3) was cultured in EMEMsupplemented with 16% heat-inactivated FCS, 2 mM L-glutamine andpenicillin (100 U/ml)/streptomycin (50 mg/ml) (Biofluids, Rockville,Md.). NS-1 and SP2/0 myeloma cells were cultured in RPMI 1640supplemented with 20% FCS, glutamine and antibiotics. C1.MC/C57.1 cellswere cultured as described in Young et al. (Proc. Natl. Acad. Sci. USA84:9175-9179 (1987)). DNP-human serum albumin (DNP-HSA) (30-40 molesDNP/mole albumin) was purchased from Sigma Chemical Co. (St. Louis,Mo.). DNP-specific IgE supernatants were used to saturate FcεRI asdescribed in Young et al. (Proc. Natl. Acad. Sci. USA 84:9175-9179(1987)). For PCA experiments, MOPC31c (IgG₁) and anti-DNP-mouse IgE(clone SPE-7) were purchased from Sigma Chemical Co. (St. Louis, Mo.)and anti-rat β2 integrin (anti-LFA-1β, CD18; clone WT.3) was purchasedfrom Pharmigen (San Diego, Calif.). MOPC 31c and anti-DNP IgE weredialyzed to remove sodium azide before in vivo injections. Anti-rat CD81(5D1, IgG₁) was purified from ascites on Protein G Sepharose (Pharmacia,Uppsala, Sweden).

Immunizations, Fusions, and FACS

Female BALB/c mice (4-8 weeks old) were immunized intraperitoneally with25×10⁶ RBL-2H3 emulsified in complete Freund's adjuvant or 50×10⁶ inPBS. Mice were boosted after 2 weeks with 40×10⁶ RBL-2H3 cellsemulsified in incomplete Freund's adjuvant intraperitoneally or in PBS.For the final immunizations, animals were injected with 20-40×10⁶RBL-2H3 cells intraperitoneally at day −4 (fusion=day 0) and intravenousat day −3. Spleen cell preparations were fused with either NS-1 or SP2/0myeloma cells in polyethylene glycol and plated onto normal BALB/cspleen feeder cells. To enhance the development of the hybridomas, S.typhimurium mitogen (Ribi ImmunoChem Research, Inc., Namilton, Mont.)was included in the culture medium from days 0-10. Hybridomasupernatants were tested after day 14 by flow cytometry for binding toRBL-2H3 using FITC-conjugated goat anti-mouse F(ab′)2-specific antibody(Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) andanalyzed by flow cytometry on a FACSCAN™ flow cytometer(Becton-Dickinson, San Jose, Calif.).

From 3 separate fusions, a total of 2160 wells were plated and 622supernatants from wells with hybridoma growth were screened by FACS forreactivity with RBL-2H3 cells. In all, 283/622 (45%) elicited detectablereactivity by FACS with membrane antigens of RBL-2H3. The screening ofRBL-2H3-reactive mAbs by serotonin release assay lead to theidentification of 1A12 (IgG_(2b)) and 5D1 (IgG₁), which werecharacterized further. Rat CD81 transfectants of were stained withpurified 1A12 and 5D1 (1 μg/10⁶ cells), counterstained with goatanti-mouse F(ab′)₂-specific antibody and analyzed by flow cytometry on aFACScan® flow cytometer.

Serotonin Release Assay and Leukotriene C4 Assays

RBL-2H3 cells were loaded with ^([3H])5-hydroxytryptamine(^([3H])serotonin; 0.1-0.3 mCi/10⁵ cells) and saturated withDNP-specific IgE in 96-well microtiter tissue culture plates (10⁵cells/well, 37° C., 5% CO₂) as described in Da'ron et al. (J. Immunol.149:1365-1373 (1992)). Monolayers were washed three times with buffer(glucose-saline, PIPES buffer (pH 7.2) containing (in mM) 25 PIPES, 110NaCl, 5 KC1, 5.6 glucose, 0.4 MgCl₂, 1 CaCl2 and 0.1% BSA), and 25 ml ofa dilution of purified antibody was added to the labeled monolayers, andplates were incubated for 30 minutes (or as indicated) at roomtemperature. Triggering of FcεRI was performed by the addition ofDNP-HSA (final concentration 10-250 ng/ml) and plates were incubated at37° C. (except as indicated in FIG. 7D) with control samples present oneach plate. Degranulation was stopped by placing the plates on ice andby the addition of 150 μl of cold culture medium per well. 100 μlaliquots were taken from replicate wells for scintillation counting.Total cellular incorporation was determined from 1% SDS/1% NP-40lysates.

Leukotriene C4 was measured from 10⁶ anti-DNP IgE saturated RBL-2H3treated with 1 μg 5DI or buffer prior to triggering with 30 ng/mlDNP-HSA. Supernatants were stored at −80° C. until measurement of LTC₄by specific enzyme immunoassay (Cayman Chemical, Ann Arbor, Mich.).

Immunoaffinity Chromatography, Electrophoresis, and Western Blotting

RBL-2H3 cells were cultured in routine culture medium in spinner flasksto a cell density of approximately 10⁶/ml, harvested by centrifugationand washed twice with cold PBS. Washed cells were extracted in 0.5 MK₂HPO₄ (pH 7.5) with proteinase inhibitors (10 μg/ml pepstatin, 5 μg/mlleupeptin, and 10 μg/ml aprotinin) at 50×10⁶/ml for 60 minutes at 4° C.with frequent mixing. N-octyglucoside (10 mM) was added during theextraction to ensure protein solubility. Post-nuclear lysates wereprepared by centrifugation at 15,000×g for 20 minutes at 4° C. Lysateswere then passed through 0.2 mM filters to remove residual debris andpassed several times over protein G-Sepharose coupled to 1A12 (2 mg/mlbed volume), washed with PBS (10 mM n-octylglucoside) and eluted with0.2 M glycine. Tris-neutralized, concentrated extracts were reduced withβ-mercaptoethanol, resolved on 12.5% preparative SDS-PAGE andtransferred to Immobilon^(SQ) (Millipore, Bedford, Mass.). The membranewas stained with amido black and the Mr 25 kDa band was excised, eluted,alkylated and digested overnight with Lys-C. Peptides were separated byreverse phase-HPLC and the peptide peak eluting at 36 minutes wassequenced. Subsequent cloning and expression of rat CD81 cDNA fromRBL-2H3 confirmed that 1A12 and 5D1 recognize rat CD81 and that CD81crosslinking inhibits FcεRI-mediated mast cell degranulation.

For anti-phosphotryosine Western blots, 0.5% Triton X-100 (BBS,proteinase inhibitors) extracts were immunoprecipitated overnight with 2μg of anti-phosphotryosine mAb 4G10 bound to protein A-Sepharose beads(4° C. with rotation). Beads were washed with lysis buffer, eluted,resolved on 12.5% SDS-PAGE, transferred to nitrocellulose membranes andimmunoblotted with 1 μg/ml 4G10 mAb, followed by incubation withHRP-conjugated anti-mouse IgG secondary antibodies and development withchemiluminescence substrates (Renaissance, Dupont/NEN, Boston, Mass.).

Construction and Screening of RBL-2H3 cDNA library in UNI-ZAP™.

Poly A+ mRNA was isolated from RBL-2H3, reverse-transcribed into cDNA,size-fractionated on Sephacryl S-500 spin columns and ligated intoUNI-ZAP-XR lambda vector according to the manufacturer's instructions(Stratagene, La Jolla, Calif.). After rescue of the cDNA inserts andappropriate restriction enzyme digests, it was determined that 96% ofthe plamids contained inserts, with an average size of 1.7 kB. 5×10⁵plaques were screened with ³²p-labeled mouse CD81 cDNA probe. Afterhybridization, nitrocellulose filters were washed once with 2×SSCcontaining 0.1% SDS (room temperature) and 3 times with 0.5×SSCcontaining 0.1% SDS at 50° C. Filters were autoradiographed and plaquespicked and eluted. Candidate plaques were subjected to three additionalrounds of plaque purification before rescue of the cDNA inserts intopBluescript. Sequencing was performed on eleven isolates and all werefound to align with accession number U19894 isolated from rat brain(Geisert, Jr., et al., Neurosci. Lett. 133:262-266 (1991); Irwin andGeisert, Jr., Neurosci. Lett. 154:57-60 (1993); Geisert, Jr., et al., J.Neurosci. 16:5478-5487 (1996)).

Transfections: Rat CD81 cDNA from two isolates was subcloned into thepBJlneo expression vector (Lin et al., Cell 85:985-995 (1996)) and 20 μgof ethanol-precipitated DNA was used for electroporation of C1.MC/C57.1cells (1050 μF, 270 v). Selection of stable transfectants was initiated48 hours later by replating at 500-10,000 cells per well with 2 mg/mlG418 (Life Technologies, Grand Island, N.Y.).

Confocal Microscopy: After overnight adherence and saturation of FcεRIwith DNP-specific IgE, RBL-2H3 cells were washed with buffer andincubated with 3 μM fluo3/AM (Molecular Probes, Eugene, Oreg.) and 0.2mg/ml Pluronic (Molecular Probes) at 37° C. for 30 minutes (5% CO₂) in abuffer containing 140 mM NaCl, 5 mM KC1, 1 mM MgCl₂, 1 mM CaCl₂, 10 mMglucose, and 1 mM Na-HEPES (pH 7.4). Dye-loaded cells were then washedonce with the same buffer before preincubation (30 minutes, roomtemperature) with buffer (±5D1, 1 μg/chamber/10⁵ cells) and triggeringwith 100 ng/ml DNP-HSA. Ca²⁺ measurements in single cells were monitoredusing a laser-scanning confocal microscope (LSM4, Zeiss, New York, N.Y.)equipped with an argon/kryton laser to excite the dye at 488 nm.Fluorescence emission above 510 nm was measured after placing a longpass filter in front of the photomultiplier tube. The confocal systemwas employed in slow scan mode and fluorescence images were collectedevery 5 seconds. Fluo-3 flourescence measurements were normalized bydividing the average flourescence intensity (F) occurring during thecourse of the experiment to the average flourescence intensitydetermined at the beginning of the experiment (F₀). All measurementswere performed at 22-24° C.

Passive Cutaneous Anaphylaxis in Rats

Male Wistar rats (275-300 g) were used in these experiments. Rats werefirst anesthetized with ether, then back skin hair was shaved and ratswere injected intradermally with 50 μl containing 100 ng anti-DNP IgE or25 ng anti-DNP-IgE mixed with 50 μg of MOPC 31c (mouse IgG₁, specificityunknown) or 5D1 (mouse IgG₁, anti-rat CD81). Control sites receivedbuffer alone (PBS containing 10 μg/ml mouse serum albumin; SigmaChemical Co., St. Louis, Mo.). Sites were marked on the skin fororientation and rats that received 100 ng anti-DNP injections received asecond injection 21 hours later with 50 μg of 5D1 or anti-rat LFA-1β(CD18; mouse IgG₁) into previously injected sites. Sites receiving IgEand IgG₁ were injected in triplicate on the same rat. Twenty-four hoursafter IgE injections, animals received 1 ml of 1 mg/ml DNP-HSAcontaining 1% Evan's Blue dye injected intravenously under etheranesthesia. Thirty minutes after intravenous injection, rats weresacrificed, and punch biopsies (2.5 cm²) were obtained, minced andextracted 3 times with hot formamide (80° C., 3 hours) (Dombrowicz etal., J. Clin. Invest. 99:915-925 (1997)). Pooled samples from tissuesites were centrifuged and absorbance at 610 nm (A₆₁₀) was measured.A₆₁₀ values were converted to μg Evan's blue based on a standard curveof dilutions of Evan's Blue in formamide.

Inhibition of Signaling Elicited Through the Low Affinity IgG ReceptorFcRγIII

RBL-2H3 cells express FcεRI, CD81 and endogenous rat FcγRIII receptors.However, no high-affinity reagent (antibody) is available to triggerthese receptors on RBL-2H3; the 2.4G2 antibody (anti-mouseFcγRII/FcγRIII) was used for this purpose. To demonstrate that CD81stimulation inhibits degranulation induced through FcγRIII signaling asit does for FcεRI, murine FcγRIIIα chain cDNA was expressed in RBL-2H3cells. FcRγ cDNA was cotransfected to assist in the surface expressionof FcγRIII complexes. In FIGS. 11A-11D, the histograms of 3 stable mouseFcγRIII RBL-2H3 transfectants are shown after staining with 2.4G2 andFITC-anti-rat IgG. Untransfected RBL-2H3 cells exhibit no detectablebinding of 2.4G2 (FIG. 11A).

FcγRIII binding of IgG is detectable only when IgG is present in theform of IgG-containing immune complexes which crosslink FcγRIIIreceptors and initiate intracellular signals. One of the methods oftriggering FcγRIII is through stimulation with crosslinked anti-FcγRIIIantibodies. In FIG. 12, RBL-2H3 and FcγRIII-transfectants of RBL-2H3were loaded with ³H-serotonin in the presence (DNP-HSA stimulation) orabsence (immune complex stimulation) of DNP-specific IgE. Afterovernight incubation, cells were washed and incubated with culture mediaor media containing 200 ng of anti-rat CD81 mAb 5D1 prior to triggeringwith optimized concentrations of DNP-HSA or with preformed immunecomplexes of 2.4G2/anti-rat IgG F(ab′)₂. Degranulation was allowed toproceed for 30 minutes at 37° C. and released ³H-serotonin wasquantitated by scintillation counting. As shown in FIG. 12, DNP-HSAinduces IgE-mediated degranulation in all four cell lines which isinhibitable by anti-CD81 mAb 5D1. 2.4G2/anti-rat IgG F(ab′)2 preformedcomplexes, but not anti-rat IgG F(ab)2 alone, induce degranulation onlyin cells expressing mFcγRIII receptors (RBL-2H3 transfectants A10, D10and H11), a process which is also inhibitable by preincubation with 5D1.This data provides the identification of CD81 as a common inhibitor ofboth FcεRI and FcγRIII.

RESULTS

5D1 mAb inhibits FcεRI-mediated degranulation by antigen. From 3separate fusions, a total of 2160 wells were plated and 622 supernatantsfrom wells with hybridoma growth were screened by FACS for reactivitywith the immunizing RBL-2H3 cells (see Table 1). In all, 283/622elicited detectable reactivity by FACS with membrane antigens ofRBL-2H3. Supernatants from the positive hybridomas were then tested forinhibition of FcεRI-mediated degranulation. RBL-2H3 cells exhibit areproducible degranulation profile to FcεRI-IgE stimulation by thecorresponding antigen DNP-HSA. Detectable serotonin release is observedwith 1 ng/ml concentrations of DNP-HSA; maximal serotonin release occurswith approximately 50 ng/ml, and at concentrations greater than 1 mg/mlDNP-HSA degranulation is inhibited, presumably because of the diminishedability of large FcεRI-IgE aggregates to signal. In FIG. 4, purified 5D1mAb inhibits IgE-mediated degranulation in RBL-2H3 cells stimulated with10, 50 or 250 ng/ml DNP-HSA, with maximal inhibition occurring at 5-20ng/10⁵ RBL-2H3 cells. RBL-2H3 cells were saturated with DNP-specific IgEand labeled with 3H-hydroxytryptamine (serotonin) 0.2 mCi/10⁵ cells/well(0.32 cm²), washed three times with triggering buffer and incubated for30 minutes at room temperature with the indicated concentration ofaffinity-purified 5D1 mAb in 25 ml total volume. After incubation, cellswere challenged with 25 μl of 2×dilution of pre-warmed DNP-HSA andtriggered for 30 minutes (37° C., 5% CO₂). Release was terminated by theaddition of 150 μl of ice-cold triggering buffer and by placing theplates on ice. 100 μl aliquots of released radioactivity as well as SDScell lysates were then harvested and scintillation counted.Degranulation-inhibitory mAb binding has little or no effect on IgE oranti-FcεRIα binding.

TABLE 1 Binding inhibition of FITC-conjugated mAbs directed to RBL-2H3surface antigens Median Fluorescence Intensity (MFI) FITC-conjugatedmAbs Preincubation Specificity 1A12 IgE 4H7 3A9 — — 37.9 75.0 83.5 289.01A12 ND 61.0 83.5 289.0 5D1  6.5 ND ND ND 4H7 rat FcεRIα 38.2  6.4  7.2 38.5 3A9 rat FcεRIα 37.5  6.5  6.8  13.4 BC4 rat FcεRIα 38.5  5.8  5.4 5.9 5.14 rat FcεRIα 39.6  5.5 84.3 161.0 AA4 ganglioside ND 12.3 59.3201.7 G_(d1b) 10⁶ RBL-2H3 cells were incubated on ice with a saturatingamount of unconjugated antibody (preincubation) for 30 minutes prior tothe addition (without washes) of a titered (subsaturating) concentrationof FITC-conjugated mAb. After washes, stained cells were analyzed byFACS and mean values histgram peaks converted to median fluorescentintensity (MFI) units.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the following claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

4 14 amino acids amino acid single linear protein 1 Phe Tyr Asp Gln AlaLeu Gln Gln Ala Val Met Xaa Asp Asp 1 5 10 14 amino acids amino acidsingle linear protein 2 Asp Tyr Asp Gln Asp Leu Gln Gln Asp Val Met XaaAsp Asp 1 5 10 14 amino acids amino acid single linear protein 3 Phe TyrAsp Gln Ala Leu Gln Gln Ala Val Met Asp Asp Asp 1 5 10 14 amino acidsamino acid single linear protein 4 Phe Tyr Asp Gln Ala Leu Gln Gln AlaVal Val Asp Asp Asp 1 5 10

We claim:
 1. An assay for identifying agents which alter CD81-modulatedsignal transduction, comprising the steps of: a) combining a cellcoexpressing CD81 and an Fc antigen receptor, wherein the Fc antigenreceptor is selected from the group consisting of: an FcεRI antigenreceptor and an FcγRIII antigen receptor, with an agent to be testedunder conditions suitable for antigen-dependent degranulation; and b)assessing degranulation by measuring the level of serotonin releasedfrom cells stimulated in the presence and absence of the agent; whereinif the level of released serotonin is altered in cells in the presenceof the agent relative to the level of serotonin released by cells in theabsence of the agent, the agent alters CD81-modulated signaltransduction.
 2. The assay of claim 1 wherein the cell coexpressing CD81and an Fc antigen receptor is selected from the group consisting of:mast cell-derived cell lines, lymphocytic cell-derived cell lines andbasophilic leukemia cell-derived cell lines.